Antibiotics 890A2 and 890A5

ABSTRACT

The antibiotics MSD 890A 2  and MSD 890A 5  and pharmaceutically acceptable salts thereof (hereinafter referred to as antibiotics 890A 2  and 890A 5 ) are active against both gram-positive and gram-negative bacteria. The antibiotics are produced by growing species of Streptomyces on suitable fermentation media.

This is a continuation of application Ser. No. 680,831, filed Apr. 28,1976, now abandoned.

BACKGROUND OF THE INVENTION

The discovery of the remarkable antibiotic properties of penicillinstimulated great interest in this field which has resulted in thefinding of many other valuable antibiotic substances such as: otherpenicillins, cephalosporins, streptomycin, bacitracin, tetracyclines,chloramphenicol, erythromycins and the like. In general theantibacterial activity of each of these antibiotics does not includecertain clinically important pathogenic bacteria. For example, some areprincipally active against only gram-positive types of bacteria.Acquired resistance over the course of widespread use of existingantibiotics in the treatment of bacterial infection has caused a seriousresistance problem to arise.

Accordingly, the deficiencies of the known antibiotics have stimulatedfurther research to find other antibiotics which will be active againsta wider range of pathogens as well as resistant strains of particularmicroorganisms.

This further research has lead to the discovery of the thienamycinfamily of antibiotics of which the compounds of the present inventionare members. Other members of the thienamycin family of antibiotics aredescribed in the applications Jean S. Kahan, Frederick M. Kahan, EdwardO. Stapley, Robert T. Goegelman and Sebastian Hernandez, U.S. Ser. No.632,938, filed Nov. 18, 1975 now U.S. Pat. No. 4,006,060 which is adivisional application of the co-pending application Jean S. Kahan,Frederick M. Kahan, Edward O. Stapley, Robert T. Goegelman and SebastianHernandez, U.S. Ser. No. 526,992, filed Nov. 25, 1974 now U.S. Pat. No.3,950,357; Robert T. Goegelman and Frederick M. Kahan, U.S. Ser. No.613,822, filed Sept. 18, 1975 now U.S. Pat. No. 4,000,161 which is acontinuation-in-part of the co-pending application Robert T. Goegelmanand Frederick M. Kahan, U.S. Ser. No. 534,382, filed Dec. 19, 1974, nowabandoned; Patrick J. Cassidy, Robert T. Goegelman, Edward O. Stapleyand Sebastian Hernandez, U.S. Ser. No. 634,300, filed Nov. 21, 1975 nowabandoned; Jean S. Kahan, Frederick M. Kahan, Robert T. Goegelman,Edward O. Stapley and Sebastian Hernandez, U.S. Ser. No. 634,301, filedNov. 21, 1975 now abandoned; and Jean S. Kahan and Frederick M. Kahan,U.S. Ser. No. 634,560, filed Nov. 24, 1975 now abandoned.

SUMMARY OF THE INVENTION

This invention is directed to new members of the thienamycin family ofantibiotic agents. More particularly, it is concerned with newantibiotic substances, herein designated 890A₂ and 890A₅. The inventionencompasses the antibiotics in dilute forms, as crude concentrates andin pure forms.

It is an object of the present invention to provide new and usefulantibiotics which are highly effective in inhibiting the growth ofvarious gram-negative and gram-positive microorganisms. Another objectis to provide a process for the preparation of these novel antibioticsubstances by the fermentation of nutrient media with species ofStreptomyces. Other objects will be apparent from the detaileddescription of this invention hereinafter provided.

The novel antibiotic substances of the present invention are produced bygrowing under controlled conditions new strains of Streptomycesflavogriseus.

Based upon extensive taxonomic studies the strains of microorganismsused in the present invention were identified as belonging to thespecies Streptomyces flavogriseus and have been designated MA-4434 andMA-4600 in the culture collection of MERCK & CO., Inc., Rahway, N.J. Aculture of each thereof has been placed on permanent deposit withoutrestrictions as to availability with the culture collection of theNorthern Regional Laboratories, Northern Utilization Research andDevelopment Division, Agricultural Research Service, U.S. Department ofAgriculture, Peoria, Ill., and are available to the public underaccession No. NRRL 8139 and 8140, respectively.

Streptomyces flavogriseus NRRL 8139 and NRRL 8140 each produce bothantibiotics 890A₂ and 890A₅ which are isolated in substantially pureform from the fermentation broth.

The morphological and cultural characteristics of Streptomycesflavogriseus NRRL 8139 are set forth in the following table.

Morphology -- Sporophores are branching, straight to flexuous chains ofspores, forming tufts. Chains are more than 10 spores in length. Sporesare spherical to oval -- 0.9μ × 1.2μ(970×).

Cultural Characteristics

Oatmeal agar

Vegetative growth -- Reverse-yellowish tan, parchment-like growth;

Aerial mycelium -- Light gray edged with medium gray

Soluble pigment -- None.

Czapek Dox agar (sucrose nitrate agar)

Vegetative growth -- Reverse-brown edged with dark brown;

Aerial mycelium -- Medium gray, velvety;

Soluble pigment -- Slight browning of medium.

Egg albumin agar

Vegetative growth -- Reverse-yellowish tan edged with brown;

Aerial mycelium -- Medium gray mixed with yellowish gray (2dc) andgrayed yellow (2db);

Soluble pigment -- Light yellowish tan.

Glycerol asparagine agar

Vegetative growth -- Reverse-yellowish tan, flat, spreading;

Aerial mycelium -- Velvety, light gray with a strong yellowish tone togray (2dc);

Soluble pigment -- None.

Inorganic salts-starch agar

Vegetative growth -- Reverse -- brown;

Aerial mycelium -- Medium gray, velvety;

Soluble pigment -- Light yellowish-tan.

Yeast extract-dextrose + salts agar

Vegetative growth -- Reverse-brown edged with very dark brown;

Aerial mycelium -- Dark gray mixed with a light gray, velvety;

Soluble pigment -- None.

Yeast extract-malt extract agar

Vegetative growth -- Reverse-dark brown;

Aerial mycelium -- Dark gray, velvety;

Soluble pigment -- None.

Skim milk agar

Vegetative growth -- Tan;

Aerial mycelium -- Sparse, grayish;

Soluble pigment -- Slight browning of medium;

Hydrolysis of casein -- Good.

Litmus milk

Vegetative growth -- Moderate growth ring, dark tan;

Aerial mycelium -- None;

Color -- Purple;

Coagulation and/or peptonization -- Complete peptonization; becomingalkaline, pH 8.2.

Skim milk

Vegetative growth -- Moderate growth ring, tan;

Aerial mycelium -- None;

Soluble pigment -- Tan;

Coagulation and/or peptonization -- Complete peptonization; becomingalkaline, pH 8.0.

Tyrosine agar

Vegetative growth -- Reverse-dark brown;

Aerial mycelium -- Dark gray;

Soluble pigment -- Slight browning of medium;

Decomposition of tyrosine -- None.

Peptone-iron-yeast extract agar

Vegetative growth -- Tan;

Aerial mycelium -- Sparse, grayish;

Soluble pigment -- None;

Melanin -- None;

H₂ s production -- None.

Nutrient agar

Vegetative growth -- Reverse-light grayish brown edged with darkergray-brown;

Aerial mycelium -- Light gray edged with dark gray;

Soluble pigment -- None.

Nutrient starch agar

Vegetative growth -- Tan edged with gray

Aerial mycelium -- Medium gray edged with dark gray;

Soluble pigment -- None;

Hydrolysis of starch -- Good

Nutrient gelatin agar

Vegetative growth -- Colorless edged with dark gray;

Aerial mycelium -- Grayish-white;

Soluble pigment -- None;

Liquefaction of gelatin -- Good.

Potato plug

Vegetative growth -- Good growth, heavily wrinkled;

Aerial mycelium -- Gray to greenish-gray;

Soluble pigment -- Slight browning of medium.

Loeffler's Blood serum

Vegetative growth -- Cream-colored;

Aerial mycelium -- None;

Soluble pigment -- None;

Liquefaction -- None.

Gelatin stabs

Vegetative growth -- Cream-colored;

Aerial mycelium -- None;

Soluble pigment -- None;

Liquefaction of gelatin -- Good.

All of the readings reported above were taken after three weeksincubation at 28° C. unless noted otherwise. The pH of the media used inthese studies was approximately neutral, namely, pH 6.8-7.2. The colordesignations used in the description are in accordance with thedefinitions of the Color Harmony Manual, 4th Edition (1958), ContainerCorporation of America, Chicago, Illinois.

Streptomyces flavogriseus NRRL 8139 was also tested for its ability toutilize or assimilate various carbohydrates. For this purpose, themicroorganism was grown on basal synthetic medium (Pridham and Gottlieb)containing 1% of the carbohydrate at 28° C. for three weeks. The pH ofthe media employed in the study was approximately neutral (6.8-7.2).Table I shows the utilization of these carbohydrate sources byStreptomyces flavogriseus NRRL 8139, + indicating good growth, ± poorgrowth, and - no growth on the particular carbohydrate.

                  TABLE 1                                                         ______________________________________                                        Glucose      +          Maltose   +                                           Arabinose    +          Mannitol  +                                           Cellulose    -          Mannose   +                                           Fructose     +          Raffinose -                                           Inositol     -          Rhamnose  +                                           Lactose      +          Sucrose   ±                                        Xylose       +                                                                ______________________________________                                    

The amount of growth with change in temperature and the oxygenrequirement by the microorganism is as follows:

Temperature range (Yeast extract-dextrose + salts agar);

28° C. -- Good

37° C. -- Good vegetative growth; no aerial hyphae

50° C. -- No growth

Oxygen requirement (Stab culture in yeast extract-dextrose + saltsagar);

Aerobic

The morphological and cultural characteristics of Streptomycesflavogriseus NRRL 8140 are set forth in the following table.

Morphology -- Sporophores are branching, straight to flexuous chains ofspores, forming tufts. Chains are more than 10 spores in length. Sporesare spherical to oval -- 0.9μ × 1.2μ(970×).

Cultural Characteristics

Oatmeal agar

Vegetative growth -- Reverse -- yellowish tan edged with dark brown;

Aerial mycelium -- Light gray edged with medium gray;

Soluble pigment -- None.

Czapek Dox agar (sucrose nitrate agar)

Vegetative growth -- Reverse -- brown edged with dark brown;

Aerial mycelium -- Medium gray, velvety;

Soluble pigment -- None.

Egg albumin agar

Vegetative growth -- Reverse -- grayish tan with sections of strongyellow tan;

Aerial mycelium -- Sections of medium gray, grayish white and yellowishgray (2dc);

Soluble pigment -- Very light tan.

Glycerol asparagine agar

Vegetative growth -- Yellowish tan;

Aerial mycelium -- Sparse, grayish;

Soluble pigment -- None.

Inorganic salts -- starch agar

Vegetative growth -- Reverse -- grayish cream;

Aerial mycelium -- Medium gray, velvety;

Soluble pigment -- None.

Yeast extract -- dextrose + salts agar

Vegetative growth -- Reverse -- dark brown;

Aerial mycelium -- Dark gray mixed with a light gray, velvety;

Soluble pigment -- None.

Yeast extract -- malt extract agar

Vegetative growth -- Reverse -- dark brown;

Aerial mycelium -- Dark gray, velvety;

Soluble pigment -- None.

Peptone -- iron -- yeast extract agar

Vegetative growth -- Tan;

Aerial mycelium -- None;

Soluble pigment -- None;

Melanin -- None;

H₂ s production -- None.

Nutrient agar

Vegetative growth -- Light tan;

Aerial mycelium -- None.

Soluble pigment -- None.

Nutrient starch agar

Vegetative growth -- Cream-colored;

Aerial mycelium -- None;

Soluble pigment -- None;

Hydrolysis of starch -- Good.

Nutrient gelatin agar

Vegetative growth -- Cream-colored;

Aerial mycelium -- None;

Soluble pigment -- None;

Liquefaction of gelatin -- Good.

Gelatin stabs

Vegetative growth -- Tan;

Aerial mycelium -- None;

Soluble pigment -- None;

Liquefaction of gelatin -- Complete.

Skim milk agar

Vegetative growth -- Tan;

Aerial mycelium -- None;

Soluble pigment -- None;

Hydrolysis of casein -- Good.

Litmus milk

Vegetative growth -- Tan growth ring;

Aerial mycelium -- None;

Color -- Brownish purple;

Coagulation and/or peptonization -- Complete peptonization, becomingalkaline, pH 8.0.

Skim milk

Vegetative growth -- Tan, moderate growth ring;

Aerial mycelium -- None;

Soluble pigment -- Light brown;

Coagulation and/or peptonization -- Complete peptonization, becomingalkaline pH 8.5.

Potato plug

Vegetative growth -- Good, tan colored;

Aerial mycelium -- Very sparse, whitish;

Soluble pigment -- None.

Loeffler's Blood serum

Vegetative growth -- Cream-colored;

Aerial mycelium -- None;

Soluble pigment -- None;

Liquefaction -- None.

Tyrosine agar

Vegetative growth -- Tan;

Aerial mycelium -- None;

Soluble pigment -- Light browning of medium;

Decomposition of tyrosine -- Very slight.

All of the readings reported above were taken after three weeksincubation at 28° C. unless noted otherwise. The pH of the media used inthese studies was approximately neutral, namely pH 6.8-7.2. The colordesignations used in the description are in accordance with thedefinitions of the Color Harmony Manual, 4th Edition (1958), ContainerCorporation of America, Chicago, Illinois.

Streptomyces flavogriseus NRRL 8140 was also tested for its ability toutilize or assimilate various carbohydrates. For this purpose, themicroorganism was grown on basal synthetic medium (Pridham and Gottlieb)containing 1% of the carbohydrate at 28° C. for three weeks. The pH ofthe media employed in the study was approximately neutral (6.8-7.2)Table II shows the utilization of these carbohydrate sources byStreptomyces flavogriseus NRRL 8140 + indicating good growth, ± poorgrowth, and - no growth on the particular carbohydrate.

                  TABLE II                                                        ______________________________________                                        Glucose      +          Maltose   +                                           Arabinose    +          Mannitol  +                                           Cellulose    -          Mannose   +                                           Fructose     +          Raffinose ±                                        Inositol     ±       Rhamnose  +                                           Lactose      +          Sucrose   ±                                        Xylose       +                                                                ______________________________________                                    

The amount of growth with change in temperature and the oxygenrequirement by the microorganism is as follows:

Temperature range (Yeast extract -- dextrose + salts agar);

28° C. -- Good

37° C. -- Moderate vegetative growth; no aerial hyphae

50° C. -- No growth

Oxygen requirement (Stab culture in yeast extract -- dextrose + saltsagar);

Aerobic

It is to be understood that for the production of new antibiotics ofthis invention, the present invention is not limited to the organism,Streptomyces flavogriseus or to organisms fully answering the abovegrowth and microscopic characteristics which are given for illustrativepurposes. In fact, it is desired and intended to include the use ofmutants produced from the described organism by various means, such asX-radiation, ultraviolet radiation, nitrogen mustard, phage exposure andthe like.

The novel antibiotics of the invention, 890A₂ and 890A₅, are producedduring the aerobic fermentation, under controlled conditions, ofsuitable aqueous nutrient media inoculated with strains of the organism,Streptomyces flavogriseus. Aqueous media, such as those employed for theproduction of other antibiotics, are suitable for producing 890A₂ and890A₅. Such media contain sources of carbon, nitrogen and inorganicsalts assimilable by the microorganism.

In general, carbohydrates such as sugars, for example, dextrose,glucose, fructose, maltose, sucrose, xylose, mannitol and the like andstarches such as dextrin or such as grains, for example, oats, rye,cornstarch, corn meal and the like can be used either alone or incombination as sources of assimilable carbon in the nutrient medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depends in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 1% and6% by weight of the medium. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium. In general, many proteinaceous materials may be used as nitrogensources in the fermentation process. Suitable nitrogen sources include,for example, yeast hydrolysates, primary yeast, soybean meal, cottonseedflour, hydrolysates of casein, corn steep liquor, distiller's solublesor tomato paste and the like. The sources of nitrogen, either alone orin combination, are used in amounts ranging from about 0.2% to 6% byweight of the aqueous medium.

Among the nutrient inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, ammonium, calcium, magnesium, phosphate, sulfate, chloride,carbonate, and like ions. Also included are trace metals such as cobalt,manganese and iron.

It should be noted that the media described in the Examples are merelyillustrative of the wide variety of media which may be employed, and arenot intended to be limitative.

The fermentation is carried out at temperatures ranging from about 20°C. to 37° C.; however, for optimum results it is preferable to conductthe fermentation at temperatures of from about 23° C. to 28° C. Theinitial pH of the nutrient media suitable for growing strains of theStreptomyces flavogriseus culture and producing antibiotics 890A₂ and890A₅ can vary from about 6.0 to 8.0.

Although the novel antibiotics 890A₂ and 890A₅ are produced by bothsurface and submerged cultures, it is preferred to carry out thefermentation in the submerged state.

A small scale fermentation of the antibiotic is conveniently carried outby inoculating a suitable nutrient medium with the antibiotic-producingculture and, after transfer to a production medium, permitting thefermentation to proceed at a constant temperature of about 28° C. on ashaker for several days.

The fermentation is initiated in a sterilized flask of nutrient mediumvia one or more stages of seed development. The nutrient medium for theseed stage may be any suitable combination of carbon and nitrogensources. The seed flask is shaken in a constant temperature chamber atabout 28° C. for one day, or until growth is satisfactory, and some ofthe resulting growth is used to inoculate either a second stage seed orthe production medium. Intermediate stage seed flasks, when used, aredeveloped in essentially the same manner; that is, part of the contentsof the flask from the last seed stage are used to inoculate theproduction medium. The inoculated flasks are shaken at a constanttemperature for several days, and at the end of the incubation periodthe contents of the flask are centrifuged or filtered.

For large scale work, it is preferable to conduct the fermentation insuitable tanks provided with an agitator and a means of aerating thefermentation medium. According to this method, the nutrient medium ismade up in the tank and sterilized by heating at temperatures of up toabout 120° C. Upon cooling, the sterilized medium is inoculated with apreviously grown seed of the producing culture, and the fermentation ispermitted to proceed for a period of time as, for example, from 1 to 6days while agitating and/or aerating the nutrient medium and maintainingthe temperature at about 24° to 28° C. This method of producingantibiotics 890A₂ and 890A₅ is particularly suited for the preparationof large quantities of the antibiotics.

Physical and chemical properties of antibiotics 890a₂ and 890a₅

properties of antibiotic 890a₂

antibiotic 890A₂ is an acidic substance which moves toward the positivepole on electrophoresis at neutral pH.

The sodium salt of antibiotic 890A₂ is a white powder as lyophilizedfrom aqueous solution, and is very soluble in water.

The ultraviolet absorbance spectrum has maxima at 308 and 228 nm, and aminimum at 262 nm. The E% at 308 nm of the sodium salt of antibiotic890A₂ in water at neutral pH is estimated to be greater than 490. Theratio of absorbance values, A₃₀₈ /A₂₆₀, is 1.91 and A₃₀₈ /A₂₂₈ is 1.02for the best samples obtained; evidence of small quantities ofimpurities remaining in these samples suggest that the ratios may beslightly higher for a sample of ultimate purity.

More than 80% of the absorption at 308 nm may be eliminated by reactionwith hydroxylamine, and a similar decrease is observed upon reactionwith cysteine. The absorbance at 260 nm after such reactions increasesby about one-fourth the magnitude of the A₃₀₈ decrease. Under theconditions described in the section headed Bioassay III for determiningthe HAEA₃₀₈ values, the reaction kinetics appear to be first order witha half-life at room temperature between 0.5 and 1.5 minutes.

The following table lists the 100 MHz-NMR signals for 890A₂ sodium saltin D₂ O relative to the internal standard, sodium2,2-dimethyl-2-silapentane-5-sulfonate, hereinafter referred to as DSS;chemical shifts are given in ppm and coupling constants in Hz; theapparent multiplicites are indicated.

1.33 (d, J=6, CH₃ --CH); 2.07 (S, CH₃ C═O);

3.15 (m, C--CH₂ --C); 3.69 (m, >CH--C═O);

˜4.3 (>CHN and >CH--OH), 6.09 and 7.12 (doublets, J=13.5, S--CH═CH--N).

The antibiotic potency of 890A₂, measured on Vibrio percolans ATCC 8461as described in the section headed Bioassay I, is approximately 180units per HAEA₃₀₈ unit.

PROPERTIES OF ANTIBIOTIC 890A₅

Antibiotic 890A₅ is an acidic substance which moves to the positive poleon electrophoresis at neutral pH.

The sodium salt is a white powder when lyophilized from aqueoussolution.

The ultraviolet absorbance spectrum has maxima at 308.5 and 228 nm, anda minimum at 262 nm. The E% at 308.5 nm of a solution of the sodium saltof antibiotic 890A₅ in water at neutral pH is estimated to be 490 for apure sample; the ratio of absorbances A₃₀₈ /A₂₆₀ is 2.0 and the ratioA₃₀₈ /A₂₂₈ is 1.03. More than 90% of the absorption at 308 nm may beeliminated by reaction with hydroxylamine, giving a product with anincreased absorbance at 260 nm. The magnitude of the A₂₆₀ increase isapproximately one-fourth of the A₃₀₈ decrease. The kinetics of the A₃₀₈decrease under the conditions described for measuring the HAEA₃₀₈ appearto be first order, with a half-life at room temperature between 1 and 3minutes.

The circular dichroism spectrum of 890A₅ has a positive maximum at 304nm with a specific ellipticity of 7189 degree-ml. per decimeter-gram, apoint of zero ellipticity at 257.5 nm, and a negative minimum at 220 nmwith a specific ellipticity of -21,218 degree-ml. per decimeter-gram. Incalculating these values, the concentration of 890A₅ is estimated fromthe absorbance at 308 nm using an E% value of 490 at that wavelength.

The following table lists the 100 MHz-NMR signals for 890A₅ sodium saltin D₂ O relative to the internal standard DSS; chemical shifts are givenin ppm and coupling constants in Hz; the apparent multiplicities areindicated.

1.29 (d, J=6.2, CH₃ --CH); 2.05 (S, CH₃ C═O);

3.09 (app. d of d, C--CH₂ --C); 3.41 (d of d, J=5.0, 3.0, >CH--C═O);4.16 (m, >CH--N and >CH--OH); 6.00 and 7.11 (doublets, J=13.8,S--CH═CH--N).

The antibiotic potency of 890A₅, measured on Vibrio percolans ATCC 8461,is 12 units per HAEA₃₀₈ unit.

Mass Spectral Analysis of 890A₂ and 890A₅

The mass spectral data for 890A₅ is obtained on trimethylsilylderivatives prepared from ammonium salts of the antibiotic withbis-trimethylsilyltrifluoro acetamide in dimethyl formamide. Conversionof the sodium salt of the antibiotic to the ammonium salts is carriedout by using the ammonium salt of an acidic ion exchange resin.

Trimethyl-silylation of 890A₂ and 890A₅ results in three differentderivatives: a di- and a tri-trimethylsilyl derivative (M.W.s 456 and528, respectively) and a small amount of a tetra-trimethylsilylderivative of a hydrolysed product (M.W. 618) wherein the β-lactam ringis open.

The values of the most important mass spectral fragments are givenbelow:

di-trimethylsilyl derivative: 441; 299; 298 and 84;

tri-trimethylsilyl derivative: 513; 371; and 156;

tetra-trimethylsilyl derivative (only low-resolution signal observed):618 and 603.

Antibiotics 890A₂ and 890A₅ are believed to be isomers having amolecular structure as follows: ##STR1##

Antibiotics 890A₂ and 890A₅ are further characterized by the followingantibiotic spectrum profiles.

The test to determine the antibiotic spectrum profile of antibiotic890A₂ is carried out by saturating 1/4 inch diameter paper discs in asolution of antibiotic 890A₂ in 5% methanol at a concentration of 5μg./ml.; air drying the saturated paper discs and placing them on thesurface of 100 × 15 mm. petri plates containing 5 ml. of seeded nutrientagar plus 0.2% yeast extract.

The test to determine the antibiotic spectrum profile of antibiotic890A₅ is carried out by application of a 0.015 ml. droplet of a 33μg./ml. aqueous solution of the antibiotic on the surface of a 100 × 15mm. petri plate containing 5 ml. of seeded nutrient agar plus 0.2% yeastextract. The results, expressed in terms of the diameter in millimetersof the zone of inhibition, are set forth in Table III.

The spectra of antibiotic 890A₂ and 890A₅ are quite similar, except forthe resistance of 890A₅ to inactivation by Difco penicillinase and bylactamase(s) produced by a cephalosporin C-resistant strain of Vibriopercolans (MB-2566). The results also indicate that antibiotic 890A₂ ismarkedly more potent than 890A₅.

                                      TABLE III                                   __________________________________________________________________________    In Vitro Antibacterial Spectrum Profile (ASP) of Antibiotics 890A.sub.2       and 890A.sub.5                                                                                          Inhib. Zone Diam., mm                               Organism      Merck No.                                                                           ATCC No.                                                                            890A.sub.2 - 5 μg./ml.                                                              890A.sub.5 - 33 μg./ml.                 __________________________________________________________________________    Bacillus sp.  MB-633                                                                              --    40       18                                         Proteus vulgaris                                                                            MB-1012                                                                             --    11       23                                         Pseudomonas aeruginosa                                                                      MB-979                                                                              --     0        0                                         Serratia marcesens                                                                          --     990  23       29                                         Staphylococcus aureus                                                                       --     6538 P                                                                             34       34                                         Bacillus subtilis                                                                           --    6633  42       27                                         Sarcina lutea --    9341  42       40                                         Staphylococcus aureus                                                                       MB-698                                                                              --    30       35                                         Streptococcus faecalis                                                                      MB-753                                                                              --    12        0                                         Alcaligenes faecalis                                                                        --     213  34       25                                         Brucella bronchiseptica                                                                     --    4617  29       22                                         Salmonella gallinarum                                                                       MB-1287                                                                             --    33       26                                         Vibrio percolans                                                                            --    8461  31       32                                         Xanthomonas vesicatoria                                                                     MB-815                                                                              --    29       20                                         Proteus vulgaris                                                                            --    21100 30       27                                         Escherichia coli                                                                            MB-1418                                                                             --    32       27                                         Pseudomonas stutzeri                                                                        --    11607 19       14                                         Klebsiella pneumoniae                                                                       MB-1264                                                                             --    31       27                                         Aerobacter aerogenes                                                                        MB-835                                                                              --    27       24                                         Erwinia atroseptica                                                                         --    4466  26       21                                         Pseudomonas aeruginosa                                                                      MB-2824                                                                             --    13        0                                         Corynebacterium pseudodiph.                                                                 --    9742  35       33                                         Escherichia coli                                                                            --    9637  29       24                                         Streptoccocus faecium                                                                       MB-2820                                                                             --    22        0                                         Streptococcus agalactiae                                                                    MB-2875                                                                             --    32       27                                         Vibrio percolans resistant                                                    to ceph. C)   MB-2566                                                                             --    11       35                                         Proteus vulgaris (episome).sup.a                                                            MB-2112                                                                             --    33       23                                         Proteus mirabilis                                                                           MB-3126                                                                             --    22       20                                         Staphylococcus aureus                                                         (res. methicillin)                                                                          MB-2949                                                                             --    11       15                                         Vibrio percolans +2 × 10.sup.5                                          units/ml. penicillinase                                                                     MB-1272                                                                             --     7       40                                         Vibrio percolans + Aerobacter                                                 Lactamase     MB-1272                                                                             --    31       36                                         __________________________________________________________________________     .sup.a this episome confers resistance to tetracycline, chloramphenicol,      kanamycin and streptomycin at a concentration of 20 μg./ml. and            neomycin at a concentration of 25 μg./ml. and sulfa drugs.            

Antibiotic 890A₂ exhibits in vivo activity against gram-negative andgram-positive organisms and hence is useful in controlling bacterialinfections in animals and humans. In determining the in vivo activity,antibiotic 890A₂ is dissolved in 0.15M NaCl, 0.01M sodium phosphate, pH7.0, and diluted with water to provide five fourfold concentrations ofdrug for testing. Female white Swiss mice, averaging about 21 g. inweight, were infected intraperitoneally with the test organism suspendedin broth. The numbers of organisms injected were determined by standardplate-count techniques. At the time of infection and again 6 hourslater, certain of the mice were treated intraperitoneally with theantibiotic. Five mice were used for each concentration of drug tested.An additional two mice, not infected, were treated with the antibioticto determine whether the amount of agent injected was toxic. Controls offive mice for each of several dilutions of the infecting culture wereincluded in each test in order to calculate the numbers of organismsthat were lethal to 50% of the infected, untreated mice (LD₅₀). Thiscalculation was made using survival data of the seventh day afterinfection, at which time the amount of drug that should protect 50% ofthe infected mice (ED₅₀) also was calculated.

All animals receiving this challenge and not treated with antibioticdied within 48 hours of the infection. The efficacy of antibiotic 890A₂,having a potency of 59 units/ml., against Salmonella schottmuellariMB-2837 is set forth below:

    ______________________________________                                                                  ED.sub.50 × 2 doses                           Units/ml. .sup.a                                                                            Route      Units                                                ______________________________________                                        59            ip         19                                                   ______________________________________                                         .sup.a 1 unit/ml. is as defined under the heading Bioassay I.            

Antibiotics 890A₂ and 890A₅ are valuable antibiotics active againstvarious gram-positive and gram-negative bacteria and, accordingly, findutility in human and veterinary medicine. The compounds of thisinvention can be used alone or in combination with each other asantibacterial drugs for treating infections caused by gram-positive orgram-negative bacteria, for example, against Staphylococcus aureus,Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae andSalmonella schottmuellari. The antibacterial materials of the inventionmay further be utilized as additives to animal feeding-stuffs, forpreserving foodstuffs and as disinfectants. For example, they may beemployed in aqueous compositions in concentrations ranging from about0.1 to about 100 parts of antibiotic per million parts of solution orpreferably in concentrations ranging from about 1 to about 10 parts ofantibiotic per million parts of solution in order to destroy and inhibitthe growth of harmful bacteria on medical and dental equipment and asbactericides in industrial applications, for example in water-basedpaints and in the white water of paper mills to inhibit the growth ofdeleterious bacteria.

The antibiotics of this invention may be used in any one of a variety ofpharmaceutical preparations as the sole active ingredients or incombination either with one or more other antibiotics or with one ormore pharmacologically active substances. As an example of the former,an aminocyclitol antibiotic such as gentamicin may be coadministered inorder to minimize any chance that resistant organisms will emerge. As anexample of the latter, diphenoxylate and atropine may be combined indosage forms intended for the therapy of gastroenteritis. Theantibiotics may be employed in capsule form or as tablets, powders orliquid solutions or as suspensions or elixirs. It may be administeredorally, topically, intravenously or intramuscularly. Furthermore theantibiotics 890A₂ and 890A₅ may be used in combination with each other.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; lubricants,for example, magnesium stearate, talc, polyethylene glycol, silica;disintegrants, for example, potato starch or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of aqueous or oily suspension, solution, emulsions, syrups,elixirs, etc. or may be presented as a dry product, for reconstitutionwith water or other suitable vehicles before use. Such liquidpreparations may contain conventional additives such as suspendingagents, for example, sorbitol syrup, methyl cellulose, glucose/sugarsyrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel or hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate or acacia; nonaqueous vehicleswhich may include edible oils, for example, almond oil, fractionatedcoconut oil, oily esters, propylene glycol, or ethyl alcohol;preservatives, for example methyl or propyl p-hydroxybenzoates or sorbicacid. Suppositories will contain conventional suppository bases, e.g.cocoa butter or other glyceride.

Compositions for injection may be presented in unit dose form inampules, or in multidose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, emulsions inoily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for reconstitution with asuitable vehicle, e.g. sterile, pyrogen-free water, before use.

The compositions may also be prepared in suitable forms for absorptionthrough the mucous membranes of the nose and throat or bronchial tissuesand may conveniently take the form of powder or liquid sprays orinhalants, lozenges, throat paints, etc. For medication of the eyes orears, the preparations may be presented as individual capsules, inliquid or semi-solid form, or may be used as drops etc. Topicalapplications may be formulated in hydrophobic or hydrophilic bases asointments, creams, lotions, paints, powders, etc.

Also, in addition to a carrier, the instant compositions may includeother ingredients such as stabilizers, binders, antioxidants,preservatives, lubricators, suspending agents, viscosity agents orflavoring agents and the like.

In veterinary medicine, such as in the treatment of chickens, cows,sheep, pigs and the like, the composition may, for example, beformulated as an intramammary preparation in either long acting orquick-release bases.

The dosage to be administered depends to a large extent upon thecondition of the subject being treated, the weight of the host and thetype of infection, the route and frequency of administration, theparenteral route being preferred for generalized infections and the oralroute for intestinal infections.

In the treatment of bacterial infections in man, the compounds of thisinvention are administered orally or parenterally, in accordance withconventional procedures for antibiotic administration, in an amount offrom about 2 to 600 mg./kg./day and preferably about 5 to 100mg./kg./day in preferably divided dosage, e.g. three to four times aday. They may be administered in dosage units containing, for example,100, 330, 400 or 1000 mg. of active ingredient with suitablephysiologically acceptable carriers or excipients. The dosage units arein the form of liquid preparations such as solutions or suspensions oras solids in tablets or capsules. It will, of course, be understood thatthe optimum dose in any given instance will depend upon the type andseverity of infection to be treated, and that smaller doses will beemployed for pediatric use, all of such adjustments being within theskill of the practitioner in the field.

Included in this invention are the non-toxic, pharmaceuticallyacceptable salts of 890A₂ and 890A₅ for example, the pharmacologicallyacceptable salts formed with inorganic and organic bases; which include,for example, metal salts derived from alkali metal or alkaline earthmetal hydroxides, carbonates or bicarbonates such as those derived fromsodium, potassium, ammonium and calcium and salts derived from primary,secondary or tertiary amines such as monoalkylamines, dialkylamines,trialkylamines, lower alkanolamines, di-loweralkanolamines, loweralkylenediamines, N,N-diaralkyl lower alkylenediamines, aralkylamines,amino substituted lower alkanols, N,N-di-lower alkylamino substitutedlower alkanols, amino-, polyamino and guanidino-substituted loweralkanoic acids and nitrogen-containing heterocyclic amines.Representative examples include salts derived from sodium hydroxide,ammonium hydroxide, sodium carbonate, sodium bicarbonate, potassiumcarbonate, potassium hydroxide, calcium carbonate, trimethylamine,triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine,lysine, protamine, arginine, procaine, ethanolamine, morphine,benzylamine, ethylenediamine, N,N'-dibenzylethylenediamine,diethanolamine, piperazine, dimethylaminoethanol,2-amino-2-methyl-1-propanol, theophylline, N-methylglucamine and thelike.

The salts of the compounds of the present invention may be prepared byconventional methods well known in the art. For example, the mono-saltssuch as monosodium salt obtained by treating one equivalent of sodiumhydroxide with one equivalent of the product (I) in a suitable solvent.Also mixed salts with divalent cations may be prepared by combining onemole of a divalent base with one mole of the product (I) plus oneequivalent of another acid. Alternatively, salts may be obtained bytreating one equivalent of a base having a divalent cation, such ascalcium hydroxide, with one equivalent of the product (I). The salts ofthis invention are pharmacologically acceptable non-toxic derivativeswhich can be used as the active ingredient in suitable unit-dosagepharmaceutical forms. Also, they may be combined with other drugs toprovide compositions having a broad spectrum of activity.

Fermentation broths containing the antibiotics 890A₂ and 890A₅ producedin accordance with the procedures described herein have activitiesranging from about 2 to 170 units per ml. when assayed in accordancewith the disc-diffusion assay using Vibrio percolans (ATCC 8461). Theantibiotics 890A₂ and 890A₅ contained in these fermentation broths canbe recovered and purified by a number of procedures. One such procedurecomprises adsorbing the antibiotics 890A₂ and 890A₅ on a strongly basicanion exchange resin. Illustrative of such strongly basic anion exchangeresins are those having a styrene-divinylbenzene matrix, for example thepolystyrene nuclear quaternary ammonium resin Dowex 1 × 2 (manufacturedby Dow Chemical Co., Midland, Michigan), on the chloride cycle. Otherrepresentative members of this class of strongly basic exchange resinsinclude the following: Duolite A-40, A-42, A-101, A-102 and A-114(manufactured by Chemical Process Co., Redwood City, California).Amberlite IRA-400, IRA-401 and IRA-410. Alternately, a weekly basicanion exchange resin such as Amberlite IRA-68 may be used. (Amberliteresins are manufactured by Rohm and Haas, Washington Square,Philadelphia 5, Pennsylvania.)

The adsorbed antibiotic is readily eluted from the anion exchange resinwith salt solutions in 50% (v/v) aqueous methanol. The eluate soobtained can be further purified, if desired, by other purificationprocedures. Thus, the eluate can be purified by concentrating it andpassing it through a column packed with an acrylic ester polymer ofintermediate polarity such as XAD-7 or 8 or through a column packed witha polystrene, non-polar, hydrophobic crosslinked divinyl benzene polymersuch as XAD-1, 2 and 4, preferably XAD-2. (XAD-1, 2, 4, 7, and 8 aremanufactured by Rohm and Haas, Washington Square, Philadelphia 5,Pennsylvania). This process partially resolves the antibiotics 890A₂ and890A₅. The fractions rich in 890A₂ and those rich in 890A₅ are pooled.These pooled fractions are further purified.

A method of obtaining further purified antibiotic 890A₂ and 890A₅ is bythe use of gel filtration through polyacrylamide gel having a pore sizewhich excludes molecules having a molecular weight greater than 1800,such as Bio-Gel P-2 (manufactured by Bio.Rad, Richmond, California).Other gels, such as Sephadex G-10 may also be employed for desalting.

The preferred procedure by which antibiotics 890A₂ and 890A₅ may beobtained in high purity from a broth consists of a centrifugation orfiltration of the broth to remove solids; an adsorption and elution ofthe filtrate from an anion-exchange resin such as Dowex-1 × 2 in thechloride cycle with 3% NaCl in 50% (v/v) aqueous methanol, which bothconcentrates and partially purifies the antibiotics; a passage over acolumn of suitably prepared XAD-2, which retards the antibiotics andthereby purifies and desalts the Dowex-1 × 2 eluate. Furthermore, theantibiotic 890A₅ is retarded more than the antibiotic 890A₂ and therebythe two antibiotics are partially resolved. The fractions enriched in890A₂ and 890A₅ are pooled and further purified. Chromatography on aDowex-1 × 2 minus 400 mesh resin, with elution by NaCl and/or NH₄ Cl in50% aqueous methanol, gives a product free from most UV-absorbingimpurities (the NH₄ Cl is used to provide some buffering capacity in theeluent); and a desalting on Bio-Gel P-2 or Sephadex G-10, removes mostof the salt introduced in the Dowex-1 × 2 chromatography.

When broths of low potency are treated by the above procedure, the finalmaterial may have significant quantities (more than 50%) of impuritiesremaining. These impurities may be reduced by an additional cycle ofchromatography on Dowex-1 × 2, minus 400 mesh, with elution by asolution containing sodium chloride and 50% isopropanol. It is alsoadvisable, when isolating antibiotic from low-potency broths, to do asecond stage of XAD-2 chromatography. This provides additionalpurification, removes residual salt, and partially resolves theantibiotic 890A₂ from 890A₅. Specifically, the 890A₅ elutes later thanthe 890A₂ and the two may be distinguished by their different ratios ofbioactivity to HAEA₃₀₈. Those fractions which demonstrate a ratio ofbioactivity on Vibrio percolans ATCC 8461 to HAEA₃₀₈ of 180 bioactivityunits per HAEA₃₀₈ unit contain antibiotic 890A₂ and those fractionswhich demonstrate a ratio of bioactivity on Vibrio percolans ATCC 8461to HAEA₃₀₈ of 12 bioactivity units per HAEA₃₀₈ unit contain antibiotic890A₅.

In purification by column chromatography, in general only thosefractions of the eluted volume which contain antibiotic at least 30% aspure as the purest fraction are combined for further purification.Criteria of purity are the ratios bioactivity/A₂₂₀, A₃₀₈ /A₂₆₀ andHAEA₃₀₈ /A₂₂₀ ; and, in desalting procedures, the conductivity. Thus ateach chromatography step the A₂₂₀, A₂₆₀, A₃₀₈ and bioactivity ofappropriate fractions are measured. Where possible, HAEA₃₀₈ is alsomeasured, and in desalting, conductivities are measured. The criteriafor deciding which fractions to combine for subsequent operations may beadjusted somewhat to achieve a higher yield, at the expense of purity,or conversely a higher purity at the expense of yield.

In laboratory-scale operations (less than 20 liters of sample volume),all chromatography steps except the XAD-2 chromatography are carried outin a cold room at 2°-5° C. The XAD-2 chromatography is carried out atroom temperature unless stated otherwise. The pH of antibiotic solutionsto be stored is adjusted to 7-8 by careful addition of dilute NaOH orHCl solutions. Aqueous solutions are stored in a refrigerator, orpreferably in ice water, and 50% methanol solutions are stored at -20°C.

At stages of purification prior to XAD-2 chromatography, solutions aregenerally brought to 25 μM in EDTA by addition of 1/4000th volume of asolution of 0.1M Na₂ EDTA which has been neutralized to pH 7.0 byaddition of sodium hydroxide (0.1M "neutral EDTA").

A flow sheet diagram of the purification procedure for obtainingantibiotics 890A₂ and 890A₅ is presented in FIGS. 1 and 2, respectively.##STR2## ASSAY PROCEDURES FOR ANTIBIOTICS 890A₂ AND 890A₅

I. Bioassay

An agar plate disc-diffusion method is employed using either Vibriopercolans ATCC 8461 or Salmonella gallinarum MB-1287 as tester organism.A purified sample of antibiotic 890A₁ is used as standard. Antibiotic890A₁ is prepared according to the procedure set forth in Example 5.

Plates containing Vibrio percolans ATCC 8461 are prepared as follows:

A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15ml. of a sterilized medium containing 8 g./liter of Difco Nutrient Brothand 2 g./liter of yeast extract in distilled water "nutrient broth-yeastextract" (herein after designated NBYE). The culture is incubatedovernight on a rotary shaker at 28° C. This culture is used to inoculatethe surface of slants containing 1.5% agar in NBYE, and the inoculatedslants are incubated overnight at 28° C., and then stored in arefrigerator.

The refrigerated slants prepared from a single lyophilized culture areused for up to four weeks from their preparation, as follows: A loop ofinoculum from the slant is dispersed in 50 ml. of NBYE contained in a250 ml. Erlenmeyer flask. The culture is incubated overnight on a rotaryshaker at 28° C. and then diluted to a density giving 50% transmittanceat 660 nm. A 33.2 ml. portion of this diluted culture is added to 1liter of NBYE containing 15 g. of agar and maintained at 46° C. Theinoculated agar-containing medium is poured into 100 × 15 mm. plasticpetri dishes, 5 ml. per dish, chilled, and maintained at 2°-4° C. for upto 5 days before using.

Plates containing Salmonella gallinarum MB-1287 are prepared as follows:

A sealed tube containing Salmonella gallinarum MB-1287 cells in skimmilk, which had been frozen and lyophilized and sealed under vacuum, isopened and inoculated into 15 ml. of Brain Heart Infusion broth. Thecells are allowed to grow without shaking at 37° C. overnight, and theculture is inoculated onto slants of 0.8% BBL Nutrient broth + 0.2%Difco yeast extract + 1.5% agar. After growth overnight at 37° C., aloop of the culture is transferred from the slant to a flask containing50 ml. of 0.8% nutrient broth + 0.2% yeast extract. The flask is shakenovernight, and 20 ml. of this culture is inoculated into one liter of0.8% nutrient broth + 0.2% yeast extract + 1.5% agar which had beensterilized and cooled to 48° C. The inoculated NBYE agar is pouredimmediately into 100 × 15 mm. plastic petri dishes, 5 ml. per dish, andthe plates are kept at 2°-4° C. until use.

Filter paper discs of one-half inch diameter are dipped into thesolution to be assayed, and are placed on the agar. Alternatively, thediscs may be loaded by pipetting one-tenth ml. of solution onto a drydisc, and then placing the disc on the agar. The diameter of the zone ofinhibition is measured after appropriate incubation (9-18 hours at 37°C. for Salmonella gallinarum MB-1287 or 12-24 hours at 25° C. for Vibriopercolans ATCC 8461.) If necessary, dilutions of the solutions to beassayed are made in 0.05M potassium phosphate buffer, pH 7.4 "potassiumphosphate buffer" (hereinafter referred to as KPB), or in deionizedwater.

Calculations of potencies proceed as follows: a slope is determined bymeasuring the zone diameters of a solution of antibiotic 890A₂ or 890A₅and of a four-fold dilution (in KPB) of this solution. Two discs of eachconcentration are assayed on a single plate, and the average zone sizeat each concentration is determined. The slope is equal to one-half ofthe difference of the average zone sizes. Potencies are then calculatedby the formula: ##EQU1## where D is the average diameter of the zonesformed by the unknown, D_(s) is the average diameter of the standardzones, and "Dilution" is the degree to which the unknown was dilutedbefore assay. If no standard is used, D_(s) is assumed to be 25 mm. and(Potency of Standard) is taken as 1 unit/ml., when measured on Vibriopercolans ATCC 8461. Pure 890A₁ is defined as having a potency of 250units per hydroxylamine-extinguishable absorbance unit a 300 nm, whenused as a standard.

For assays on Salmonella gallinarum MB-1287 plates, a slope isdetermined in the same way as on Vibrio percolans ATCC 8461 plates. Whenno standard is used, only relative potencies are calculated. If no slopeis measured, a value of 2.3 mm. is assumed. The potency calculationsproceed as with the Vibrio percolans ATCC 8461 assay. If no 890A₁standard is used, a control of penicillin G at 250 μg/ml. may beemployed in order to verify that the sensitivity of the organisms is inthe normal range.

II. Assay Procedure for Determining "890 Assay Units"

A conventional agar plate disc-diffusion method is employed using Vibriopercolans ATCC 8461 as tester organism. Cephaloridine is employed as astandard. Plates containing Vibrio percolans ATCC 8461 are prepared asfollows. A culture of Vibrio percolans ATCC 8461 is incubated innutrient broth-yeast extract overnight on a rotary shaker at 28° C. andthen diluted to a density of 60% transmittance at 660 nm. A 33.2 ml.portion of this diluted culture is added to 1 liter of a medium composedof nutrient agar plus 0.2% yeast extract maintained at 46° C. Theinoculated agar-containing medium is poured into 100 × 15 mm.plastic-petri dishes, 10 ml. per dish, chilled, and maintained at 2°-4°C. for up to 5 days before use.

The concentration of cephaloridine which is equivalent to 1 unit/ml. of890A is deterined by assay on plates prepared as above, but containing 5ml. of inoculated medium per plate, as follows. Four concentrations ofcephaloridine constitute the standard -- 3.12, 6.25, 12.5 and 25 mcg perml. with the 12.5 mcg per ml. as a reference solution. The zonediameters on a 5 ml. plate for the standard are as follows:

    ______________________________________                                        Conc. (mcg./ml.)  Zone Diameter (mm.)                                         ______________________________________                                        3.12              16.8                                                        6.25              22.3                                                        12.5              25.0                                                        25                29.6                                                        ______________________________________                                    

A unit is defined as the amount of antibiotic per ml. producing a 25 mm.zone of inhibition on a 5 ml. plate as described in section I above.Therefore, in this assay a concentration of 2.5 mcg per ml. ofcephaloridine is considered equivalent to 1 unit of 890A per ml. Sincethe slope of the line for cephaloridine is 4.0 calculations of thepotency of a sample are made using a slope of 4.0.

III. Hydroxylamine Reaction

Both antibiotics 890A₂ and 890A₅ react with hydroxylamine and produce asubstance with greatly diminished absorbance at 308 nm. This providesthe basis for a quantitative assay of the antibiotics 890A₂ and 890A₅.

The solution to be assayed is brought to 0.05M in potassium phosphate,pH 7.4 by adding 1/20th volume of a solution containing 0.8M K₂ HPO₄ and0.2M KH₂ PO₄. Then one-hundredth volume of 1M hydroxylaminehydrochloride is added, and the absorbance at 308 nm is measured atintervals of one-half to two minutes. The reaction is conducted at roomtemperature. First-order kinetics are assumed and a half-life isestimated from the absorbance decrease during the first ten minutes.From this half-life, the time is estimated beyond which no furtherabsorbance decrease should be observed and observations are continuedbeyond that time. If no further decrease is observed beyond that time,the total absorbance decrease (correcting for dilution effect andabsorbance of the hydroxylamine) is taken as the"Hydroxylamine-extinguishable absorbance at 308 nm (HAEA₃₀₈)". Ifabsorbance decrease is observed beyond that time, the rate of backgroundabsorbance decrease is calculated, and the observed decrease at thattime is corrected for background decrease, assuming that backgrounddecrease is linear with time. The corrected value is then recorded asthe HAEA₃₀₈.

The number of HAEA₃₀₈ units is equal to the HAEA₃₀₈ multiplied by thevolume in ml.

The examples which follow illustrate the methods by which the productsof this invention may be obtained. However, the examples areillustrative only and it should be apparent to one having ordinary skillin the art that this invention includes the functionally equivalentproducts and methods for their preparation. Therefore, any modificationof the processes described herein which results in the formation of theproducts of this invention should be construed as constituting ananalogous method. The described processes are capable of wide variationand modification and any minor departure or extension is considered asbeing within the skill of the artisan and as falling within the scope ofthis invention.

EXAMPLE 1

Production of Mixture Containing Antibiotic 890A₂ and 890A₅

A tube of lyophilized culture of Streptomyces flavogriseus MA-4434 isopened aseptically and the contents suspended in a tube containing 0.8ml. sterile Davis salts solution having the following composition:

Davis Salts

Sodium citrate -- 0.5 g.

K₂ HPO₄ -- 7.0 g.

KH₂ PO₄ -- 3.0 g.

(NH₄)₂ SO₄ -- 1.0 g.

MgSO₄.7H₂ O -- 0.1 g.

Distilled H₂ O -- 1000 ml.

This suspension is used to inoculate 4 slants of sterile medium A havingthe following composition:

    ______________________________________                                        Medium A                                                                      ______________________________________                                        Dextrose              10.0     g.                                             Peptone               5.0      g.                                             Yeast Extract         3.0      g.                                             NaCl                  12.705   g.                                             KCl                   0.72     g.                                             FeSO.sub.4 (NH.sub.4).sub.2 SO.sub.4 . 6H.sub.2 O                                                   0.0351   g.                                             MgCl.sub.2 . 6H.sub.2 O                                                                             5.32     g.                                             CaCl.sub.2 . 2H.sub.2 O                                                                             0.728    g.                                             Distilled H.sub.2 O   1000     ml.                                            pH 7.4 (before sterilization)                                                 Agar                  25.0     g.                                             ______________________________________                                    

The inoculated slants are incubated for one week at 27°-28° C. and thenstored at 4°-6° C. until used 10 days later. A one-half portion of thesurface growth of one of these slants is used to inoculate baffled 250ml. Erlenmeyer flasks containing 50 ml. of medium B having the followingcomposition:

    ______________________________________                                        Medium B                                                                      Yeast Autolysate (.sup.+ Ardamine)                                                                    10.0      g.                                          Glucose                 10.0      g.                                          Phosphate Buffer*       2.0       ml.                                         MgSO.sub.4 . 7H.sub.2 O 50        mg.                                         Distilled H.sub.2 O     1000      ml.                                          pH: adjust to 6.5 using HCl or NaOH                                          .sup.+ Ardamine: Yeast Products Corporation                                   *Phosphate Buffer solution                                                    KH.sub.2 PO.sub.4       91.0      g.                                          Na.sub.2 HPO.sub.4      95.0      g.                                          Distilled H.sub.2 O     1000      ml                                          ______________________________________                                    

Three seed flasks are inoculated to provide sufficient inoculum for the12 two liter production flasks fermented in this batch. The seed flasksare shaken for one day at 28° C. on a 220 rpm shaker (2" throw). Theflasks are removed from the shaker and stored for one day at 4° C. Thecontents of these seed flasks are used to inoculate 12 two literunbaffled Erlenmeyer production flasks (7 ml. of inoculum per flask)containing 250 ml. of the production medium C having the followingcomposition:

    ______________________________________                                        Medium C                                                                      ______________________________________                                        Tomato Paste          20.0     g.                                             Primary Yeast         10.0     g.                                             Dextrin (Amidex)      20.0     g.                                             Deionized H.sub.2 O   1000     ml.                                            pH adjust to 7.3 using NaOH                                                   ______________________________________                                    

After inoculation, production flasks are incubated at 24° C. withagitation on a 220 rpm shaker (2" throw) for four days and five hours.At the end of this period flasks are harvested and assayed for activityby using standard Salmonella gallinarum MB1287 and Vibrio percolans ATCC8461 assay plates using 1/2 inch assay discs dipped into centrifugedbroth samples. The results are tabulated below:

    ______________________________________                                        Assay at Harvest                                                              ______________________________________                                        Harvest age (hours)     101                                                   pH                      7.1                                                   Salmonella gallinarum                                                         (mm. zone)              31                                                    Vibrio percolans                                                              (mm. zone)              43                                                    ______________________________________                                    

The fermentation broth is centrifuged. To the clear centrifugate, 2.2l., at pH 6.8, is added 22 mg. of (ethylenedinitrilo)tetraacetic acidand the pH is adjusted to 7.2. The pH adjusted centrifugate is adsorbedon 150 ml. of Dowex 1 × 2 resin in the Cl⁻ cycle at 15 ml./min.collecting the spent stream as one fraction. The adsorbate is washedwith 150 ml. of deionized water containing 10 μg./ml. of(ethylenedinitrilo) tetraacetic acid and then washed with 5% NaClcontaining 10 μg./ml. of (ethylenedinitrilo) tetraacetic acid collecting5 × 75 ml. fractions.

The 5% NaCl eluting solution is followed by a 60 ml. displacement washof deionized water and then the antibiotics 890A₂ and 890A₅ and elutedwith 90% (v/v) methanol; 3% ammonium chloride (w/v/); 10% water (v/v)containing 10 μg./ml. (ethylenedinitrilo) tetraacetic acid, collecting 5× 75 ml. fractions. Fractions were assayed against Vibrio percolans,ATCC 8461 and the results are tabulated below as percent of startingbioactivity:

    ______________________________________                                        Fraction                Recovery                                              ______________________________________                                        Feed                    100     %                                             Spent                   0                                                     Water displacement wash 1       %                                             MeOH/NH.sub.4 Cl eluates fractions                                            1 to 3                  24      %                                             ______________________________________                                    

EXAMPLE 2

Shake Flask Production of Antiobiotic 890A₂

A tube of lyophilized culture of Streptomyces flavogriseus MA-4434 isopened aseptically and the contents suspended in a tube containing 0.8ml. of sterile Davis salts having the following composition:

Davis Salts

Sodium citrate -- 0.5 g.

K₂ HPO₄ -- 7.0 g.

KH₂ PO₄ -- 3.0 g.

(NH₄)₂ SO -- 1.0 g.

MgSO₄.7H₂ O -- 0.1 g.

Distilled H₂ O -- 1000 ml.

This suspension is used to inoculate four slants of sterile medium Ahaving the following composition:

    ______________________________________                                        Medium A                                                                      ______________________________________                                        Glycerol              20.0     g.                                             Primary Yeast         5.0      g.                                             Fish Meal             15.0     g.                                             Distilled H.sub.2 O   1000     ml.                                            Agar                  20.0     g.                                             pH: adjust to 7.2 using NaOH                                                  ______________________________________                                    

The inoculated slants are incubated for one week at 27°-28° C. and thenstored at 4°-6° C. until used (not longer than 21 days).

A one-third portion of the growth from one slant is used to inoculateone baffled 250 ml. Erlenmeyer flask. A total of four slants are used toinoculate twelve flasks. Each flask contains 50 ml. of medium B havingthe following composition:

    ______________________________________                                        Medium B                                                                      Yeast Autolysate (.sup.+ Ardamine)                                                                    10.0      g.                                          Glucose                 10.0      g.                                          Phosphate Buffer*       2.0       ml.                                         MgSO.sub.4 . 7H.sub.2 O 50        mg.                                         Distilled H.sub.2 O     1000      ml.                                          pH: adjust to 6.5 using HCl or NaOH                                          .sup.+ Ardamine: Yeast Products Corporation                                   *Phosphate Buffer solution                                                    KH.sub.2 PO.sub.4       91.0      g.                                          Na.sub.2 HPO.sub.4      95.0      g.                                          Distilled H.sub.2 O     1000      ml                                          ______________________________________                                    

The seed flasks are shaken for one day at 27°-28° C. on a 220 rpm shaker(2" throw). The flasks and contents are stored stationary for one day at4° C.

Forty-four 2 liter Erlenmeyer production flasks, each containing 200 ml.of medium C, are inoculated with 8 ml. per flask of the growth from theseed flasks. The medium C has the following composition:

    ______________________________________                                        Medium C                                                                      ______________________________________                                        Tomato Paste           20.0     g.                                            Primary Yeast          10.0     g.                                            Dextrin (Amidex)       20.0     g.                                            CoCl.sub.2 . 6H.sub.2 O                                                                              5.0      mg.                                           Distilled H.sub.2 O    1000     ml.                                           pH: adjust to 7.2-7.4 using NaOH                                              ______________________________________                                    

After inoculation, the production flasks are incubated at 24° C., withagitation on a 212 rpm shaker (2" throw), for four days and five hours.The flasks are harvested and assayed for activity by using standardSalmonella gallinarum MB1287 and Vibrio percolans ATCC 8461 assay platesusing 1/2 inch assay discs dipped into centrifuged broth samples.Samples are diluted with 0.02M phosphate buffer, pH 7.0 when necessary.The results are tabulated below:

    ______________________________________                                        Harvest Age Hours        101                                                  ______________________________________                                        pH                      7.2                                                   Salmonella gallinarum                                                         (mm zone)               35                                                    Vibrio percolans 1/10                                                         dil (mm zone)           25                                                    890 Assay Units         121                                                   ______________________________________                                    

The total of 7.0 liters of whole broth obtained from this fermentationis chilled to 3° C. and centrifuged in 200 ml. portions at 9000 rpm for15 minutes each. To the combined supernatant is added 1.7 ml. of 0.1Mneutral EDTA and the batch is held at 3° C.

The above fermentation is repeated under identical conditions with theexception that the 44 two liter Erlenmeyer production flasks areinoculated with 7 ml. per flask with growth from the seed flasks. The pHand assay results are tabulated below:

    ______________________________________                                        Harvest Age Hours       101                                                   ______________________________________                                                                7.3                                                   Salmonella gallinarum                                                         (mm zone)               38                                                    Vibrio percolans, 1/10                                                        dil (mm zone)           27                                                    890 Assay Units         92.8                                                  ______________________________________                                    

The total of 7.4 liters of whole broth obtained from this fermentationis chilled to 3° C. and centrifuged in 200 ml. portions at 9000 rpm for15 minutes each. To the combined supernatant is added 1.8 ml. of 0.1Mneutral EDTA.

The supernatants from the centrifuged broths resulting from the twoabove fermentations in this Example are combined to give a total volumeof 13 liters and a potency of 80 units/ml. by assay on Vibrio percolansATCC 8461.

The combined supernatant is passed through a column of Dowex-1 × 2(Cl⁻), 50-100 mesh, with bed dimensions 4.7 cm. × 50 cm., at a flow rateof 60 ml. per minute. The column is washed with 1 liter of deionizedwater, and is eluted with 3 liters of 5% (w/v) NaCl solution containing0.01M Tris-HCl buffer, pH 7.0, and 25μM neutral EDTA, at a flow rate of50 ml. per minute.

The column is washed with 500 ml. of deionized water, and theantibiotics 890A₂ and 890A₅ are eluted with 2 liters of 3% NaCl + 0.01MTris-HCl, pH 7.0, + 25μM neutral EDTA in 50% methanol, at a flow rate of40 ml./minute. Fractions of 220 L ml. are collected.

Antibiotic activity, as judged by assay on Salmonella gallinarum MB1287,appears in fractions 4 through 8, with a maximum in fraction 5.Fractions 4 through 8 contain 9% of the initial activity present inbroth.

Fractions 4 through 7 are combined, concentrated to 150 ml. by rotaryevaporation under reduced pressure, and diluted to 350 ml. by additionof 200 ml. of deionized water. The sample is applied to a column (5 cm.× 25 cm.) of XAD-2, which had been washed previously with 3 liters eachof 60% (v/v) acetone, deionized water and 5% (w/v) NaCl. The column iseluted with 500 ml. of deionized water at 20 ml./minute followed by 500ml. of 60% (v/v) aqueous acetone. Fractions of 160 ml. each arecollected. Antibiotic activity, determined by assay on Salmonellagallinarum MB1287, appears in fractions 1 through 5, with a maximum infractions 3 and 4.

Fraction 4, the first acetone-containing fraction is concentrated to 10ml. under reduced pressure, and diluted with 90 ml. of deionized water.This sample is applied to a column (5 cm. × 25 cm.) of XAD-2, which hadbeen washed previously with 3 liters each of 60% (v/v) acetone,deionized water and 5% (w/v/) NaCl. The column is eluted with deionizedwater at a flow rate of 20 ml. per minute. Fractions of from 140 to 190ml. are collected. Antibiotic activity, determined by assay onSalmonella gallinarum MB1287, appears in fractions 2 through 7 with amaximum at fraction 4. Fractions 3, 4 and 5 are combined and added tofraction 3 from the XAD column described in the previous paragraph. Thecombined fractions contain 26,400 bioassay units, determined by assay onVibrio percolans ATCC 8461, equal to 2.5% of the starting activity.

The combined XAD-2 fractions 3, from the first XAD column, and fractions3, 4 and 5 from the second XAD column are concentrated under reducedpressure to 25 ml. and then 25 ml. of deionized water and 50 ml. ofmethanol are added. The methanolic solution is adsorbed to a column(2.15 × 26.4 cm.) of Dowex-1 × 4 (Cl⁻), minus 400 mesh, which had beenequilibrated with 50% aqueous methanol (v/v). The column is eluted with1.44 liters of 0.07M NaCl + 0.005M NH₄ Cl + 0.0001M NH₃ in 50% methanol,at a flow rate of 2 ml. per minute. Fractions of 12.1 ml. are collected.The elution is continued with 2 liters of 0.08M NaCl + 0.005M NH₄ Cl +0.0001M NH₃ in 50% methanol, collecting fractions of 11.7 ml. each.

A peak of UV absorbance at 300 nm appears in fractions 138 through 162,with a maximum at fractions 149-150. The absorption at 305 nm isobserved to be extinguishable to the extent of 77% by reaction withL-cysteine at neutral pH. Fractions 145 through 155, containingantibiotic 890A₂, are pooled and concentrated to 2.4 ml., containing atotal of 70.5 A₃₀₀ units.

The concentrated sample is applied to a column (2.2 × 70 cm.) of Bio-GelP-2, 200-400 mesh, which had been washed with one liter of deionizedwater. The sample is allowed to drain to bed level, and the residue iswashed in with two rinses of 1 ml. each of deionized water. The columnis eluted with deionized water at the rate of 1 ml. per minute.Fractions of 2 to 3.5 ml. are collected.

The main peak of absorbance at 308 nm appears in fractions 64 to 72,immediately preceding and not completely resolved from the sodiumchloride. Fractions 66 and 67 are combined, containing 27 absorptionunits of antibiotic 890A₂ at 308 nm and from 29 to 43 mg. of NaCl (asestimated by conductivity). These combined fractions were concentratedto 1.5 ml. under reduced pressure and lyophilized, giving 43.7 mg. ofsolids.

EXAMPLE 3

Preparation of Antibiotics 890A₂ and 890A₅

A tube of lyophilized culture of Streptomyces flavogriseus MA-4434 isopened aseptically and the contents suspended in a tube containing 0.8ml. of sterile Davis salts having the following composition:

Davis Salts

Sodium citrate -- 0.5 g.

K₂ HPO₄ -- 7.0 g.

KH₂ PO₄ -- 3.0 g.

(NH₄)₂ SO₄ -- 1.0 g.

MgSO₄.7H₂ O -- 0.1 g.

Distilled H₂ O -- 1000 ml.

This suspension is used to inoculate four slants of medium A having thefollowing composition:

    ______________________________________                                        Medium A                                                                      ______________________________________                                        Glycerol              20.0     g.                                             Primary Yeast         5.0      g.                                             Fish Meal             15.0     g.                                             Distilled H.sub.2 O   1000     ml.                                            Agar                  20.0     g.                                             pH: adjust to 7.2 using NaOH                                                  ______________________________________                                    

The inoculated slants are incubated for one week at 27°-28° C. and thenstored at 4°-6° C. until used (not longer than 21 days).

Ten ml. of medium B having the composition:

    ______________________________________                                        Medium B                                                                      ______________________________________                                        Yeast Autolysate (.sup.+ Ardanine)                                                                    10.0     g.                                           Glucose                 10.0     g.                                           Phosphate Buffer*       2.0      ml.                                          MgSO.sub.4 . 7H.sub.2 O 50       mg.                                          Distilled H.sub.2 O     1000     ml.                                          pH: adjust to 6.5 using HCl or NaOH                                           .sup.+ Ardamine: Yeast Products Corporation                                   *Phosphate Buffer solution                                                    KH.sub.2 PO.sub.4       91.0     g.                                           Na.sub.2 HPO.sub.4      95.0     g.                                           Distilled H.sub.2 O     1000     ml.                                          ______________________________________                                    

is transferred aseptically to one of these slants, the spores and aerialmycelia scraped into suspension, and 3.3 ml. of this suspension used toinoculate a 2 liter baffled Erlenmeyer flask containing 500 ml. ofMedium B. This seed flask is shaken at 28° C. on a 160 rpm shaker (2"throw) for 36 hours at which time the growth is satisfactory.

The growth from this seed flask is used to inoculate a 189 literstainless steel seed tank containing 160 liters of Medium B. This tankis operated at 28° C. using an agitation rate of 150 rpm and an airflowof 3 cu. ft. per minute for 24 hours. Defoamer, Polyglycol 2000 (DowChemical Corp.), is used as required but not to exceed 0.1%. pHdeterminations are made as follows:

    ______________________________________                                        Age, Hours        0          12                                               ______________________________________                                        pH                6.3        6.35                                             ______________________________________                                    

Forty-three liters of the growth in this seed tank is used to inoculatea 756 liter stainless steel fermentor containing 467 liters of Medium C,wherein Medium C has the composition:

    ______________________________________                                        Medium C                                                                      ______________________________________                                        Tomato Paste           20.0     g.                                            Primary Yeast          10.0     g.                                            Dextrin (Amidex)       20.0     g.                                            CoCl.sub.2 . 6H.sub.2 O                                                                              5.0      mg.                                           Distilled H.sub.2 O    1000     ml.                                           pH: adjust to 7.2-7.4 using NaOH                                              ______________________________________                                    

This tank is run at 25° C. using an agitation rate of 146 rpm and anairflow of 9 cu. ft. per minute for 92 hours. Additional defoamer,Polyglycol 2000, is added as required, not to exceed 0.1%. Antibacterialassays are run on Salmonella gallinarum MB1287, Vibrio percolans ATCC8461 and the data is as follows:

    __________________________________________________________________________    Age Hrs.   0 12 24 36 48 60 72 84  92                                         __________________________________________________________________________    pH         6.6                                                                             6.7                                                                              6.65                                                                             6.3                                                                              6.0                                                                              6.4                                                                              6.15                                                                             6.5 6.5                                        MB-1287 mm.                                                                              --                                                                              -- -- S  -- 19 26 28  32                                         ATCC 8461 mm.(1-10)                                                                      --                                                                              --    S  -- 21 24 26  30                                         890A units/ml.     NA NA 6.8                                                                              13.5                                                                             24.9                                                                              24.3                                       __________________________________________________________________________

The 890A units/ml. are determined as set forth in the section with theheading "II. Assay Procedure for Determining `890 Assay Units`".

One-hundred-twenty-five gallons of broth are chilled to 5° C. andcentrifuged through a Titan P-9 centrifuge. Fifty pounds of Celite isadded to the 100 gallons of supernatant, and the suspension filteredthrough a Shriver 18-inch filter press. The 91 gallons of filtrate isadsorbed to a column containing seven gallons of Dowex-1 × 2 (Cl⁻),50-100 mesh, and the column is washed with 10 gallons of deionizedwater, followed by thirty gallons of 5% NaCl + 0.01M Tris-HCl buffer, pH7.0 + 25μM neutral EDTA in deionized water. After an additional wash of5 gallons of deionized water, the antibiotics 890A₂ and 890A₅ are elutedwith twenty-five gallons of 3% NaCl + 0.01M Tris-HCl buffer, pH 7.0 +25μM neutral EDTA in 50% (v/v) aqueous methanol. Five gallon fractionsare collected.

Antibiotic activity determined by assay against Salmonella gallinarumMB1287 appears in fractions 1 through 5 with a maximum at fraction 3.Fractions 2 and 3, containing 10.9% of the activity of the appliedbroth, are combined and concentrated to 2 gallons under reducedpressure. The concentrated sample is diluted with 8 gallons of deionizedwater and again concentrated to 2 gallons under reduced pressure, toeliminate residual methanol.

The two gallons of concentrate are applied to a column containing 10gallons of XAD-2 which had been previously washed with 50 gallons of 60%aqueous acetone followed by 50 gallons of deionized water and 50 gallonsof 5% NaCl. The column is eluted with 37.5 gallons of deionized water.Three fractions of 2.5 gallons followed by six fractions of five gallonsare collected. The activity, as determined by assay on Salmonellagallinarum MB1287 plates, appears in fractions 1 through 6, with a peakof potency in fraction 2. Fractions 2 through 5, containing 64% of theactivity applied to the XAD column or 520,000 units, are pooled.

Fractions 2 through 5 are concentrated to 120 ml. by evaporation underreduced pressure. The pH is adjusted to 6.5 and the concentrate isapplied to a column (7 × 50 cm.) of XAD-2 which had been washed with 8liters of 60% aqueous acetone followed by 4 liters of deionized waterand 8 liters of 5% NaCl in deionized water. The sample is drained to bedlevel and the column is rinsed with three 20 ml. portions of deionizedwater, draining to bed level each time. The antibiotic is eluted withsix liters of deionized water at a flow rate of 40 ml. per minute. Eightfractions of 200 ml. followed by eleven fractions of 400 ml. arecollected. Antibiotic activity, as determined by assay on Salmonellagallinarum MB1287 plates, appears in fractions 3 through 18, with a peakof activity in fraction 8.

Fractions 8 through 12, having the highest ratios of bioactivity/A₂₂₀,and having 225,000 bioactivity units, determined by assay againstSalmonella gallinarum MB1287, are combined for further processing.

The combined fractions are concentrated under reduced pressure to 50ml., and 50 ml. of methanol are added. The sample is applied on a columnof Dowex-1 × 4 (Cl⁻), minus 400 mesh, bed dimensions 2.2 × 40 cm., whichhad been previously washed with 50% methanol. The column is eluted withtwo liters of 0.07M NaCl + 0.005M NH₄ Cl + 0.001M NH₃ in 50% aqueousmethanol, at a flow rate of 2 ml. per minute. Fractions of 12 ml. arecollected.

Two unequal peaks of bioactivity, as determined by assay againstSalmonella gallinarum MB1287, are observed in the effluent,corresponding to two peaks of material with an absorption maximum at 308nm. Most of the absorbance at 308 nm in both peaks is extinguishable byreaction with hydroxylamine.

The first peak, containing 890A₂, appears in fractions 98 through 136,and the second peak, containing 890A₅, appears in fractions 136 through158. Fractions 112 through 130 are combined to give the 890A₂ Dowexpool, and fractions 138 through 148 are combined to give the 890A₅ Dowexpool. The 890A₂ is further purified by desalting on Sephadex G-10.

Fractions 112 through 130 containing 890A₂, are concentrated underreduced pressure to 5.5 ml. and the concentrate is applied to a column(2.15 × 70 cm.) of Sephadex G-10, and eluted with deionized water at aflow rate of 1 ml. per minute. Fractions of 2.85 ml. each are collected.The bioactivity as determined by assay against Salmonella gallinarumMB1287 appears in fractions 60 through 98. Fractions 78 through 90 havethe highest A₃₀₈ /A₂₆₀ ratios, and these are pooled for lyophilization.The pH of the pooled fractions is 4.1, and is adjusted to 7.8 byaddition of 8 μl of 1M ammonia. The pool contains 52 A₃₀₈ units with anA₃₀₈ /A₂₆₀ ratio of 0.76, demonstrating substantial degradation of theantibiotic during this procedure.

The pooled fractions 78 through 90 are concentrated to 1.69 ml. underreduced pressure. Two 0.1 ml. samples are removed and lyophilizedseparately in small conical glass reaction vials. The remainder islyophilized in a 14 ml. glass screw-cap vial, giving 1.29 mg. of solids,containing 890A₂.

EXAMPLE 4

Preparation of 890A₅

A tube of lyophilized culture of Streptomyces flavogriseus MA-4434 isopened aseptically and the contents suspended in a tube containing 0.8ml. of sterile Davis salts having the following composition:

Davis Salts

Sodium citrate -- 0.5 g.

K₂ HPO₄ -- 7.0 g.

KH₂ PO₄ -- 3.0 g.

(NH₄)₂ SO₄ -- 1.0 g.

MgSO₄.7H₂ O -- 0.1 g.

Distilled H₂ O -- 1000 ml.

This suspension is used to inoculate four slants of medium A having thefollowing composition:

    ______________________________________                                        Medium A                                                                      ______________________________________                                        Glycerol              20.0     g.                                             Primary Yeast         5.0      g.                                             Fish Meal             15.0     g.                                             Distilled H.sub.2 O   1000     ml.                                            Agar                  20.0     g.                                             pH: adjust to 7.2 using NaOH                                                  ______________________________________                                    

The inoculated slants are incubated for one week at 27°-28° C. and thenstored at 4°-6° C. until used (not longer than 21 days).

Ten ml. of medium B having the composition:

    ______________________________________                                        Medium B                                                                      ______________________________________                                        Yeast Autolysate (.sup.+ Ardanine)                                                                    10.0     g.                                           Glucose                 10.0     g.                                           Phosphate Buffer*       2.0      ml.                                          MgSO.sub.4 . 7H.sub.2 O 50       mg.                                          Distilled H.sub.2 O     1000     ml.                                          pH: adjust to 6.5 using HCl or NaOH                                           .sup.+ Ardamine: Yeast Products Corporation                                   *Phosphate Buffer solution                                                    KH.sub.2 PO.sub.4       91.0     g.                                           Na.sub.2 HPO.sub.4      95.0     g.                                           Distilled H.sub.2 O     1000     ml.                                          ______________________________________                                    

is transferred aseptically to one of these slants, the spores and aerialmycelia scraped into suspension, and this suspension used to inoculatethree 2 liter baffled Erlenmeyer flask containing 500 ml. of Medium B tothe extent of 3.3 ml./flask. These seed flasks are shaken at 28° C. on a160 rpm shaker (2" throw) for 36 hours at which time the growth issatisfactory.

Five hundred ml. of the growth from these seed flasks is used toinoculate a 189 liter stainless steel seed tank containing 160 liters ofMedium B. This tank is operated at 28° C. using an agitation rate of 150rpm and an airflow of 3 cu. ft. per minute for 24 hours. Defoamer,Polyglycol 2000 (Dow Chemical Corp.), is used as required but not toexceed 0.1%. pH determinations are made as follows:

    ______________________________________                                        Age, Hours        0          24                                               ______________________________________                                        pH                6.1        5.6                                              ______________________________________                                    

Forty-three liters of the growth in this seed tank is used to inoculatea 756 liter stainless steel fermentor containing 460 liters of Medium C,wherein Medium C has the composition:

    ______________________________________                                        Medium C                                                                      ______________________________________                                        Tomato Paste           20.0     g.                                            Primary Yeast          10.0     g.                                            Dextrin (Amidex)       20.0     g.                                            CoCl.sub.2 . 6H.sub.2 O                                                                              5.0      mg.                                           Distilled H.sub.2 O    1000     ml.                                           pH: adjust to 7.2-7.4 using NaOH                                              ______________________________________                                    

which had been sterilized 60 minutes at 120° C. This tank is run at 25°C. using an agitation rate of 160 rpm and an airflow of 10 cu. ft. perminute for 144 hours. Additional defoamer, Polyglycol 2000, is added asrequired, not to exceed 0.1%. Antibacterial assays are run on Salmonellacallinarum MB1287, Vibrio percolans ATCC 8461 and the data is asfollows:

    ______________________________________                                        Age in Hours                                                                              0     24     48   72   96   120  144                              ______________________________________                                        pH          6.2   6.2    6.3  7.0  7.3  7.7  8.2                              MB1287 mm   --    T      26.5 28   33.5 32.5 31.5                             MB1272 mm                                                                     (1-10 dil.) --    O      21.5 26.5 35   31.5 33                               890A units/ml.                                                                            --    NA     6.4  16.5 16.7 22.5 19.5                             ______________________________________                                    

The 890A units/ml. are determined as set forth in the section with theheading "II. Assay Procedure for Determining `890 Assay Units`".

One hundred gallons of broth are chilled to 5° C. and centrifugedthrough a Titan P-9 centrifuge. Forty pounds of Celite are added to the75 gallons of supernatant, and the suspension filtered through a Shriver18-inch filter press. The 60 gallons of filtrate are adsorbed to acolumn containing seven gallons of Dowex-1 × 2 (Cl⁻), 16-100 mesh, andthe column is washed with ten gallons of deionized water followed bythirty gallons of 5% NaCl + 0.01M Tris-HCl buffer, pH 7.0 + 25μM neutralEDTA. After an additional wash of 5 gallons of deionized water, theantibiotics 890A₂ and 890A₅ are eluted with thirty-five gallons of 3%NaCl + 0.01M Tris-HCl, pH 7.0 + 25μM neutral EDTA in 50% methanol.Fractions of five gallons each are collected.

The bioactivity, determined by assay against Salmonella gallinarumMB1287, appears in fractions 1 through 7 with a maximum at fraction 3.

Fractions 3 through 7, containing 18% of the applied activity, areconcentrated under reduced pressure to 2.5 gallons and the concentrateis applied to a column containing 10 gallons of XAD-2 which had beenpreviously washed with 50 gallons each of 60% (v/v) aqueous acetone,deionized water, and 5% NaCl in deionized water. The antibiotics 890A₂and 890A₅ are eluted with 35 gallons of 25μM neutral EDTA in deionizedwater. One fraction of 5 gallons followed by four fractions of 2.5gallons and four fractions of 5 gallons are collected. Bioactivity,determined by assay against Salmonella gallinarum MB1287, appears infractions 3 through 9, with a maximum at fraction 4.

Fractions 3 through 6, containing 18% of the activity of the filteredbroth, are combined and concentrated under reduced pressure to 122 ml.,and the concentrate is adjusted to pH 6.45 by addition of 5 ml. of 1MHCl. The concentrate is applied on a column (7.9 × 60 cm.) of XAD-2which had been previously washed with 16 liters each of 60% aqueousacetone, deionized water, and 5% NaCl in deionized water. Theantibiotics 890A₂ and 890A₅ are eluted with 8 liters of deionized waterat a flow rate of 50 ml. per minute. Fractions of from 200 to 1000 ml.are collected.

Bioactivity, determined by assay against Salmonella gallinarum MB1287,appears in fractions 5 through 20, being from 1900 to 7700 ml. of elutedvolume. The A₂₂₀, and HAEA₃₀₄ values are also measured on fractions 9through 20. Fractions 9 through 12 have 261-286 biopotency units perHAEA₃₀₄ unit, and fractions 16 through 20 have 23-28 units/HAEA₃₀₄ unit,and fractions 13 to 16 have intermediate values, demonstrating asignificant separation of antibiotics 890A₂ and 890A₅ on this column.Fractions 15 through 19, containing mostly 890A₅, are pooled for furtherpurification.

The pooled fractions, 15 through 19, are concentrated under reducedpressure to 250 ml. To the concentrate are added 250 ml. of methanol,and the sample applied at a flow rate of 2 ml. per minute to a column(3.4 × 50 cm.) of Dowex-1 × 4 (Cl⁻), minus 400 mesh, which had beenwashed with 50% methanol. The column is eluted with 8 liters of 0.1MNaCl + 0.005M NH₄ Cl + 0.0001M NH₃ in 50% methanol at a flow rate of 2ml. per minute. Fractions of 10 ml. are collected.

Antibiotic 890A₅ elutes in fractions 505 through 605 with a peak atfraction 550. Fractions 520 through 580 are pooled, and a 300 ml.portion of the pool is removed and concentrated to 4 ml. under reducedpressure. To the concentrate is added 6 ml. of methanol and 25 μl. of 1MNaOH, giving a final pH of 7.3.

The pH-adjusted concentrate is applied on a column (2.2 × 70 cm.) ofSephadex G-10 which had been equilibrated with 0.02 mM NH₃ in 50%methanol. The column is eluted with 0.02 mM NH₃ in 50% methanol at aflow rate of 1 ml. per minute.

The 890A₅ elutes in fractions 38 through 88 with a maximum at fractions39 and 40. Fractions 40 through 68 are combined and the pH adjusted from6.3 to 6.8 with 1M NaOH. The pooled fractions, 40 through 68, areconcentrated to 4 ml. under reduced pressure, and 80 μl. of 1M NaOH areadded during the concentration to maintain the pH between 7.0 and 7.3.To the final 4 ml. of concentrate, 20 μl. of 1M NaOH are added, bringingthe pH to 7.4.

The concentrate is further purified by applying on a column (3.4 × 46cm.) of Dowex-50 × 2 (Na⁺), 200-400 mesh, which had been washed with 4liters of 0.2 mM NaOH followed by 800 ml. of deionized water. Theantibiotic is eluted with deionized water at a flow rate of 4.1 ml. perminute. Fractions of 8.2 ml. are collected.

The antibiotic appears in fractions 22 through 27 with a maximum atfraction 23. Fractions 23 through 25 are pooled, containing 152 A₃₀₈units with a ratio A₃₀₈ /A₂₆₀ of 2.00. Conductivity measurementsindicate the presence of 70 μmoles of NaCl in the pooled fractions.

A 4 ml. portion of the pooled Dowex-50 fractions 23-25 is converted tothe ammonium form by passage over a column (0.7 cm. × 12 cm.) ofDowex-50 × 2 (NH₄ ⁺), 200-400 mesh, which had been washed with 100 ml.of 0.1 mM NH₃ followed by 10 ml. of deionized water. The sample iseluted with deionized water, collecting fractions of 1 to 2 ml. Allfractions with A₃₀₈ greater than 1 are combined, giving a total of 6 ml.with A₃₀₈ = 30. This sample is concentrated under reduced pressure to0.725 ml., and two 50 μl. aliquots are pipetted separately into 2conical 0.5 ml. reaction vials and lyophilized.

Trimethyl-silylation of antibiotic 890A₅ results in three differentderivatives: a di- and a tri-methylsilyl derivative (M.W.s 456 and 528,respectively) and a small amount of a tetra-trimethylsilyl derivative ofa hydrolysed product (M.W. 618) wherein the β-lactam ring is open.

The values of the most important mass spectral fragment are given below:

di-trimethylsilyl derivative: 441; 299; 298 and 84;

tri-trimethylsilyl derivative: 513; 371 and 156;

tetra-trimethylsilyl derivative (only low-resolution signal observed):618 and 603.

A 18.6 ml. portion of the pooled Dowex-50 fractions 23-25 isconcentrated to 3 ml. under reduced pressure, and a 1.96 ml. aliquot isfrozen and lyophilized in a 14 ml. glass vial to give 2.73 mg. of solidscontaining a mixture of antibiotic 890A₅ and sodium chloride. Since theconductivities of fractions 23-25 indicated that 45% of the solidsconsisted of sodium chloride, an E% of 490 at 308 nm can be calculatedfor a salt-free sample of 890A₅, from the observed E% of 272. The 1.0ml. remaining from the 3.0 ml. of concentrate is lyophilized separatelyfor NMR analysis.

The following table lists the 100 MHz-NMR signals for 890A₅ sodium saltin D₂ O relative to the internal standard DSS; chemical shifts are givenin ppm and coupling constants in Hz; the apparent multiplicites areindicated.

1.29 (d, J = 6.2, CH₃ --CH); 2.05 (S, CH₃ C═O); 3.09 (app. d of d,C--CH₂ --C); 3.41 (d of d, J = 5.0, 3.0, >CH--C═O); 4.16 (m, >CH--Nand >CH--OH); 6.00 and 7.11 (doublets, J = 13.8, S--CH═CH--N).

EXAMPLE 5

Shake Flask Production of Antibiotic 890A₁

A tube of lyophilized culture of Streptomyces flavogriseus MA-4600 isaseptically opened and the contents suspended in a tube containing 1.5ml. of sterile medium A having the following composition:

    ______________________________________                                        Medium A                                                                      Yeast Extract         10.0     g.                                             Glucose               10.0     g.                                             MgSO.sub.4 . 7H.sub.2 O                                                                             0.05     g.                                             *Phosphate Buffer     2        ml.                                            Distilled H.sub.2 O   1000     ml.                                            *Phosphate Buffer solution                                                    KH.sub.2 PO.sub.4     91.0     g.                                             Na.sub.2 HPO.sub.4    95.0     g.                                             Distilled H.sub.2 O   1000     ml.                                            ______________________________________                                    

This suspension is used to inoculate a 250 ml. triple-baffled Erlenmeyerseed flask containing 54 ml. of seed medium B having the followingcomposition:

    ______________________________________                                         Medium B                                                                     Autolyzed Yeast (Ardamine.sup.+)                                                                      10.0     g.                                           Glucose                 10.0     g.                                           MgSO.sub.4 . 7H.sub.2 O 0.05     g.                                           *Phosphate Buffer       2        ml.                                          Distilled H.sub.2 O     1000     ml.                                           pH: adjust to 6.5 with NaOH                                                  .sup.+ Ardamine: Yeast Products Corporation                                   *Phosphate Buffer solution                                                    KH.sub.2 PO.sub.4       91.0     g.                                           Na.sub.2 HPO.sub.4      95.0     g.                                           Distilled H.sub.2 O     1000     ml.                                          ______________________________________                                    

The seed flask is stoppered with cotton and shaken for 30 hours at 28°C. ± 1° C. on a 220 rpm gyrotory shaker (2" throw).

Fifty 250 ml. unbaffled Erlenmeyer production flasks, each containing 40ml. of production medium C are inoculated with 1 ml. per flask of thebroth from the seed flask. The production flasks are stoppered withcotton.

    ______________________________________                                        Medium C                                                                      ______________________________________                                        Tomato Paste           20.0     g.                                            Primary Yeast          10.0     g.                                            Dextrin (Amidex)       20.0     g.                                            CoCl.sub.2 . 6H.sub.2 O                                                                              5.0      mg.                                           Distilled H.sub.2 O    1000     ml.                                           pH: adjust to 7.2-7.4 using NaOH                                              ______________________________________                                    

After inoculation, the production flasks are incubated at 28° C. ± 1° C.with shaking on a 220 rpm gyrotory shaker (2" throw) for 3 days. Theflasks are assayed for activity against standard Vibrio percolans ATCC8461 assay plates using 1/2 inch assay discs dipped into centrifugedfermentation broth samples. Samples are diluted with 0.05M phosphatebuffer, pH 7.4. The results are tabulated below:

    ______________________________________                                        Harvest Age hours     72                                                      ______________________________________                                        pH                    6.4                                                     Vibrio percolans                                                              (1/100 Dilution) Assay                                                                              23      mm.                                             890 Assay, units/ml.  103                                                     ______________________________________                                    

The 890A units/ml. are determined as set forth in the section with theheading "II. Assay Procedure for Determining `890 Assay Units`".

The whole broth is centrifuged in 200 ml. portions in polycarbonatebottles at 9000 rpm for 15 minutes to give 1600 ml. of combinedsupernatants with a potency of 104 units/ml. To this is added 0.5 ml. of0.1M neutral EDTA.

The centrifuged broth is adsorbed on a Dowex-1 × 2 (Cl⁻), 50-100 meshcolumn, bed dimensions 3.8 × 22 cm., at a flow rate of 6 to 20 ml./min.The column is rinsed with 100 ml. of deionized water and eluted with 1liter of deionized water containing 50 g. of sodium chloride, 0.02M TrisHCl buffer, pH 7.0, and 25 μM neutral EDTA, at a flow rate of 6 ml./min.Fractions of 10 ml. are collected.

Antibiotic 890A₁ appears in fractions 13 through 81, with a maximum atfractions 25 to 33, counting from the first application of salt eluate.Fractions 24 through 41, having the highest biopotency/A₂₂₀ ratios, arecombined for further processing. The combined fractions have a total of29,000 units, or 17% of the applied bioactivity.

The Dowex eluate is concentrated to 10 ml., the pH is adjusted to 6.5with dilute hydrochloric acid, and the concentrate is applied on acolumn of XAD-2, bed dimensions 3.3 × 36 cm., which had been previouslywashed with 2 liters each of 60% aqueous acetone, deionized water, and5% (w/v) sodium chloride in deionized water. The sample is eluted withdeionized water at a flow rate of 6 ml./min. Fractions of 40 to 260 ml.are collected.

Antibiotic activity appears in fractions 6 through 14, extending from220 to 2560 ml. of eluted volume. The peak is at fractions 9 and 10,extending from 370 to 590 ml. of eluted volume. Fractions 9 through 12,extending from 370 to 1060 ml. of eluted volume, have the highest ratiosof HAEA₃₀₀ /A₂₂₀, and are combined for further processing. Thesefractions have 36,600 units, equal to 126% of the apparent appliedactivity.

The combined fractions 9 through 12 are concentrated to 100 ml. and theconcentrate applied on a column of Dowex-1 × 4 (Cl⁻), minus 400 mesh,bed dimensions 2.2 × 41 cm., at a flow rate of 2 ml./min. The column isrinsed with 50 ml. of deionized water, and eluted with 3 liters of 0.07MNaCl + 0.005M NH₄ Cl + 0.0001M NH₃ is deionized water, at a rate of 2ml./min. Fractions of 10.8 ml. are collected, starting from the firstapplication of eluent.

The main peak of antibiotic 890A₁ appears in fractions 181 through 217,with a maximum at fraction 198. Fractions 186 through 210, containing atotal of 114 absorption units at 300 nm., are pooled.

The pooled fractions are concentrated to 4.0 ml., and the pH is adjustedto 7.3 by addition of 16μ liter of 1M NaOH. The concentrate is appliedon a column of Bio-Gel P-2, 200-400 mesh, bed dimension 2.15 × 70 cm.,and is washed in with 3 × 1 ml. washes of deionized water and elutedwith deionized water at 0.96 ml./min. Fractions of 3.85 ml. arecollected.

The main peak of antibiotic 890A₁ appears in fractions 24 through 44,with a maximum at fractions 33 and 34. Fractions 27 through 38, havingthe highest A₃₀₀ /A₂₄₅ ratios, are combined for lyophilization. Thesecombined fractions have a total of 72 A₃₀₀ units.

To carry out the lyophilization, the combined fractions are concentratedto 3.0 ml. and the pH of the concentrate is adjusted to 7.5 by additionof 10μ liters of 0.1M NaOH. The sample is divided into two portions of1.50 ml. each, and the portions are separately quick-frozen andlyophilized from 14 ml. glass screw-cap vials. Each sample contains 1.73mg. of 890A₁, corresponding to 35.8 A₃₀₀ units.

Formulation containing antibiotics 890a₂ and 890a₅

compositions containing the antibiotics of the present invention may beadministered in several unit dosage forms as, for example, in solid orliquid orally ingestible dosage form. It will be apparent that theantibiotics 890A₂ and 890A₅ can be used separately or in combination.The compositions described below apply to antibiotics 890A₂ and 890A₅alone and in combination. The quantities of antibiotic 890A₅administered will, in general, be larger than the quantities of 890A₂for any given clinical situation. The compositions per unit dosage,whether liquid or solid may contain from 0.1% to 99% of active material,the preferred range being from about 10 to 60%. The composition willgenerally contain from about 100 mg. to about 2000 mg. by weight of theactive ingredient based upon the total weight of the composition;however, in general, it is preferable to employ a dosage amount in therange of from about 250 mg. to 2000 mg. In parenteral administration theunit dosage is usually the pure compound in a sterile water solution orin the form of a soluble powder intended for solution. Representativeformulations can be prepared by the following procedures:

    ______________________________________                                        Capsules                 Per Capsule                                          ______________________________________                                        Antibiotic 890A.sub.2    400 mg.                                              Lactose, U.S.P., a sufficient quantity to                                     fill No. 0 Capsules, approx. 475 mg. each                                     ______________________________________                                    

In the above example the active compound and the diluent are mixed toproduce a uniform blend, which is then filled into No. 0 hard gelatincapsules, by hand or on a suitable machine, as required. The mixing andfilling is preferably done in an area having a relative humidity lessthan 40%.

    ______________________________________                                        Tablets               Per Tablet                                              ______________________________________                                        Antibiotic 890A.sub.2 330.    mg.                                             Calcium phosphate     192.    mg.                                             Lactose, U.S.P.       190.    mg.                                             Cornstarch            80.     mg.                                             Magnesium stearate    8.      mg.                                                                   800.    mg.                                             ______________________________________                                    

In the above example, the active component is blended with the calciumphosphate, lactose and about half of the cornstarch. The mixture isgranulated with a 15% cornstarch paste and rough-screened and screenedagain through No. 16 screens. The balance of the cornstarch and themagnesium stearate is added and the mixture is compressed into tablets,approximately 1/2" in diameter, each weighing 800 mg.

Alternatively, the active component is blended with the calciumphosphate, lactose and one-half the cornstarch. The mixture is "slugged"on a heavy duty press to produce compacted tablet-like masses. These arebroken down to a No. 16 mesh granule. The balance of the cornstarch andthe magnesium stearate are added and the mixture is compressed intotablets approximately 1/2" in diameter, each weighing 800 mg.

    ______________________________________                                        Lyo Form (For Injection)                                                                              Per Vial                                              ______________________________________                                        Antibiotic 890A.sub.2   500     mg.                                           Water-for-Injection, U.S.P. to make                                                                   2       ml.                                           ______________________________________                                    

In the above example the active component is dissolved in sufficientwater-for-injection in the ratio shown. The solution is filtered throughSelas candles or Millipore membrane filters to sterilize. The solutionis subdivided into sterile vials. The vials and contents are frozen, andthe water is aseptically removed by lyophilization. The vials containingthe sterile dry solid are aseptically sealed.

To restore for parenteral administration, 2 ml. of sterilewater-for-injection is added to the contents of a vial.

    ______________________________________                                        Oral Liquid Forms      Per 1000 ml.                                           ______________________________________                                        Antibiotic 890A.sub.2  1.0      gm.                                           Sucrose                600.0    gm.                                           Glucose                250.0    gm.                                           Sodium Benzoate        1.0      gm.                                           Concentrated Orange Oil                                                                              0.2      ml.                                           Purified water U.S.P. to make                                                                        1000.0   ml.                                           ______________________________________                                    

The sucrose and glucose are dissolved in about 400 ml. of water usingheat to aid solution. This solution is cooled and sodium benzoate,followed by the concentrated orange oil added. The solution is broughtto about 900 ml. volume with water and the antibiotic is added. Thesolution is clarified by filtration through a coarse filter.

What is claimed is:
 1. The compound having the structure: ##STR3##wherein said compound has 100 MHz nuclear magnetic resonance signalshaving chemical shifts in parts per million and multiplicities indicatedas: 1.33 (d, J = 6, CH₃ --CH); 2.07 (S, CH₃ C═O); 3.15 (m, C--CH₂ --C);3.69 (m, >CH--C═O); ˜4.3 (>CHN and >CH--OH), 6.09 and 7.12 (doublets, J= 13.5, S--CH═CH--N); and the pharmaceutically acceptable salts thereof.2. The compound having the structure: ##STR4## wherein said compound has100 MHz nuclear magnetic resonance signals having chemical shifts inparts per million and multiplicities indicated as: 1.29 (d, J = 6.2, CH₃--CH); 2.05 (S, CH₃ C═O); 3.09 (d of d C--CH₂ --C); 3.41 (d of d, J =5.0, 3.0 >CH--C═O); 4.16 (m, >CH--N and >CH--OH); 6.00 and 7.11(doublets, J = 13.8, S--CH═CH--N); and the pharmaceutically acceptablesalts thereof.
 3. The mixture comprising the compound having thestructure: ##STR5## wherein said compound has 100 MHz nuclear magneticresonance signals having chemical shifts in parts per million andmultiplicities indicated as: 1.33 (d, J = 6, CH₃ --CH); 2.07 (S, CH₃C═O); 3.15 (m, C--CH₂ --C); 3.69 (m, >CH--C═O); ˜4.3 (>CHN and >CH--OH),6.09 and 7.12 (doublets, J = 13.5, S--CH═CH--N) and the compound havingthe structure: ##STR6## wherein said compound has 100 MHz nuclearmagnetic resonance signals having chemical shifts in parts per millionand multiplicities indicated as: 1.29 (d, J = 6.2, CH₃ --CH); 2.05 (S,CH₃ C═O); 3.09 (d of d C--CH₂ --C); 3.41 (d of d, J = 5.0,3.0 >CH--C═O); 4.16 (m, >CH--N and >CH--OH); 6.00 and 7.11 (doublets, J= 13.8, S--CH═CH--N).
 4. A composition comprising an antibacterialeffective amount of the compound having the structure: ##STR7## whereinsaid compound has 100 MHz nuclear magnetic resonance signals havingchemical shifts in parts per million and multiplicities indicated as:1.33 (d, J = 6, CH₃ --CH); 2.07 (S, CH₃ C═O); 3.15 (m, C--CH₂ --C); 3.69(m, >CH--C═O); ˜4.3 (>CHN and >CH--OH), 6.09 and 7.12 (doublets, J =13.5, S--CH═CH--N) and pharmaceutically acceptable salts thereof and anon-toxic pharmaceutically acceptable carrier therefore.
 5. Acomposition comprising an antibacterial effective amount of the compoundhaving the structure: ##STR8## wherein said compound has 100 MHz nuclearmagnetic resonance signals having chemical shifts in parts per millionand multiplicities indicated as: 1.29 (d, J = 6.2, CH₃ --CH); 2.05 (S,CH₃ C═O); 3.09 (d of d C--CH₂ --C); 3.41 (d of d, J = 5.0,3.0 >CH--C═O); 4.16 (m, >CH--N and >CH--OH); 6.00 and 7.11 (doublets, J= 13.8, S--CH═CH--N).
 6. A composition comprising an antibacterialeffective amount of the compound having the structure: ##STR9## whereinsaid compound has 100 MHz nuclear magnetic resonance signals havingchemical shifts in parts per million and multiplicities indicated as:1.33 (d, J = 6, CH₃ --CH); 2.07 (S, CH₃ C═O); 3.15 (m, C--CH₂ --C); 3.69(m, >CH--C═O); ˜4.3 (>CHN and >CH--OH), 6.09 and 7.12 (doublets, J =13.5, S--CH═CH--N) in combination with the compound having thestructure: ##STR10## wherein said compound has 100 MHz nuclear magneticresonance signals having chemical shifts in parts per million andmultiplicities indicated as: 1.29 (d, J = 6.2, CH₃ --CH); 2.05 (S, CH₃C═O); 3.09 (d of d C--CH₂ --C); 3.41 (d of d, J = 5.0, 3.0 >CH--C═O);4.16 (m, >CH--N and >CH--OH); 6.00 and 7.11 (doublets, J = 13.8,S--CH═CH--N) and pharmaceutically acceptable salts thereof and anon-toxic pharmaceutically acceptable carrier therefore.